Method and apparatus for using an electromagnetic field in cellular transplantation

ABSTRACT

An electromagnetic field is applied to transplanted cells during their transplantation culturing to achieve confluence and alignment of transplanted cells into damaged tissue and organs, therefore improving the synchronization of electrical and mechanical tissue or organ functions. The electromagnetic field may be applied by catheter based devices to a selected portion of tissue or to the entire surface of a hollow body cavity or organ, such as a heart chamber. The electromagnetic field is provided by an expansible net or array from a catheter or on an inflatable balloon or stent. The electromagnetic field is also provided by an implantable net or array of electrodes which may either be hardwired to a pulse generator or coupled by means of wireless or inductive transmission.

RELATED APPLICATIONS

The present application is related to U.S. Provisional PatentApplication Ser. No. 60/366,705, filed on Mar. 22, 2002, which isincorporated herein by reference and to which priority is claimedpursuant to 35 USC 119.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to the field of cellular transplantation in livingorganisms.

2. Description of the Prior Art

Transplantation of somatic cells to supply the function of a deficientorgan has been studied for decades. This technology, however, has onlybeen successfully performed for bone marrow in clinical practice. Recentstudies in skeletal muscles for Duchenne muscular dystrophy, in theliver for a bridge to transplantation, in the pancreas for islets ofLangerhans or in the brain have also shown some inconsistent results.

Cell transplantation in the heart, also called cellular cardiomyoplasty,was most intensely studied, since it may be become a useful tool forrepairing injured heart tissue. Like skeletal muscle, transplantation ofcontractile cells is required for improving the function of the injuredheart. Like brain cells, adult cardiac myocytes are terminallydifferentiated. Most strategies used to attempt to overcome thisdisadvantage involves transplanting cells which have the ability tomultiply. Among all kinds of cells, cultured fetal cardiomyocytes,allogeneic fetal skeletal myoblasts, especially, cultured autologousmyoblasts have been shown to have some functional benefits, such asdecreased infarct size and increased ejection fraction by 20%-30%.However, the results are again inconsistent. Most recently, severalhuman trials have been initiated in Europe and the first case has beenperformed in U.S.

The major problem addressed by this technique is that the arrangement oftransplanted cells usually is disorganized. These transplanted cells arenot able to align with the surrounding cells and are rarely shown to behistologically confluent with recipients' myocytes. Electrical andmechanical non-synchronization between transplanted cells andrecipient's tissue remains the major limitation for efficacy in clinicalapplication.

BRIEF SUMMARY OF THE INVENTION

The illustrated embodiment of the invention is directed to the conceptand applicable methodology for using an electromagnetic field to improvethe confluence, alignment and electrical and mechanical synchronizationof transplanted cells in the heart, skeletal muscle and nerve. A newlydesigned electromagnetic device is used for cardiac myocyte, skeletalmuscle cell and neuron cell transplantation in a hollow organ, such asthe heart and vessel, or in tissue, such as skeletal muscle and nervefibers. This invention contemplates both: 1) a method of applying anelectromagnetic field for cell transplantation; and 2) a device forapplying of an electromagnetic field for cell transplantation in heart.

While the apparatus and method has or will be described for the sake ofgrammatical fluidity with functional explanations, it is to be expresslyunderstood that the claims, unless expressly formulated under 35 USC112, are not to be construed as necessarily limited in any way by theconstruction of “means” or “steps” limitations, but are to be accordedthe full scope of the meaning and equivalents of the definition providedby the claims under the judicial doctrine of equivalents, and in thecase where the claims are expressly formulated under 35 USC 112 are tobe accorded full statutory equivalents under 35 USC 112. The inventioncan be better visualized by turning now to the following drawingswherein like elements are referenced by like numerals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is a side elevational view of a human heart showing theinjection of transplanted cells into a chamber or target tissue areawhich has been identified by conventional means as damaged.

FIG. 1 b is a side elevational view of a human heart of FIG. 1 a showingthe implantation or positioning of an electrode net or array coupled toa pulse generator for creating a predetermined electrical field duringpredetermined phases of cellular transplantation.

FIG. 2 a illustrates a catheter-based means for providingelectromagnetic manipulation of predetermined phases of cellulartransplantation in a portion of target tissue after intracoronaryinjection of cells.

FIG. 2 b illustrates a catheter-based means for providingelectromagnetic manipulation of predetermined phases of cellulartransplantation in a whole chamber of a hollow organ, such as a heart.

FIG. 2 c is a simplified cross-sectional view in enlarged scale of thefringing electromagnetic field which is impressed into adjacent orproximal tissue by the electrode nets or arrays of the invention.

FIG. 2 d illustrates a catheter-based means for providingelectromagnetic manipulation of predetermined phases of cellulartransplantation in an elongate hollow chamber, such as a vessel.

FIG. 2 e is a simplified side view of the catheter-based means of FIG. 2d after the stent has been expanded in the vessel.

FIG. 2 f is a simplified cross-sectional view in enlarged scale of thefringing electrical field which is impressed into adjacent or proximaltissue of the vessel by the electrode nets or arrays provided in theexpansible stent.

FIG. 3 is a simplified side view of a remotely powered, implanted net orarray used to provide an oriented electromagnetic field during repeatedor long term cellular transplantation, such as a receiving arrayimplanted into a selected portion of a heart wall. This electrode arrayenables us to predetermine the orientation of transplanted cell growingwith any angles or curves which will be coaligned in the direction ofthe surrounding tissue.

FIGS. 4 a and 4 b are diagrammatic depictions of devices and/or arraysimplanted over, onto or into the heart in a whole-organ manner in FIG. 4a or relative to a selected region as shown in FIG. 4 b.

FIG. 5 a is a pair of microphotographs, the left one of which shows aneonatal cardiac myocyte culture grown without EMF stimulation and theright one of which shows a neonatal cardiac myocyte culture grown withEMF stimulation.

FIG. 5 b is a photograph of the measurement of connexin 43 protein inneonatal cardiac myocyte cultures grown with EMF in the case of samples1-3 and without EMF stimulation in the case of samples 4-6.

FIG. 6 a shows segments of data showing the effect of EMF stimulation onthe Ca++ transient in neonatal cardiac myocytes in culture. The datasegment on the left is the Ca++ transient for cells grown without EMFstimulation, and the data segment on the right is the Ca++ transient forcells grown with EMF stimulation.

FIG. 6 b is a bar graph showing the average amplitude of cell motion inμm as a function of electrical field strength to which the cells havebeen exposed during growth according to the invention.

FIG. 7 is a microphotograph showing the confluence between old cellgrowth in culture and newly seeded cells in the culture when EMFstimulation according to the invention is applied during growth.

FIG. 8 is a microphotograph showing the change in orientation of cellgrowth in culture when the direction of the field of the appliedelectrical stimulation is changed or different on different sides of theanode, depicted by a solid line in the center of the microphotograph.

The invention and its various embodiments can now be better understoodby turning to the following detailed description of the preferredembodiments which are presented as illustrated examples of the inventiondefined in the claims. It is expressly understood that the invention asdefined by the claims may be broader than the illustrated embodimentsdescribed below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The concept of the invention is to introduce a new strategy, namely theapplication of an electromagnetic field for achieving confluence andalignment of transplanted cells in damaged tissue and organs, thereforeimproving the synchronization of electrical and mechanical tissue ororgan functions.

Our preliminary studies have shown that the arrangement of any culturedfetal or adult cardiac myocytes, smooth muscle cells and neuron cellsgrown in vitro are disorganized compared with those in native tissue. Ifa pulsed electromagnetic field is applied to the cells during theirculture, all of the cells grow along with the direction of theelectromagnetic field. A peak electrical field strength of 50 mV/cm to5V/cm is effective to induce a preferred or oriented growth pattern ofthe cells. A low frequency pulse rate of 0.2 Hz to 20 Hz has beenobserved to be efficacious to orient growth patterns. However, it mustbe expressly understood that many different field strengths and pulserates, or field patterns may be employed other than those depicted anddescribed here without departing from the spirit and scope of theinvention.

Our preliminary data have shown that with an electromagnetic fieldstimulation (5 ms pulse, 1 Hz, 5 V/cm), the proliferation of culturedcardiac myocytes was significantly increased. The cell number inneonatal cardiac myocytes cultures was increased 3.5 fold compared withthose without electric field stimulation as shown in the right side ofmicrophotograph of FIG. 5 a as compared to the left side respectively.The arrangement of the cells becomes organized. The gap-junction currentis increased and the expression level of Connexin-43 is significantlyincreased as shown in EMF samples 1-3 on the left side of FIG. 5 b ascompared to the non-EMF samples 4-6 on the right side of FIG. 5 b.Whole-cell Ca++ current as depicted in the graph of FIG. 6 a, Ca++transient and the amplitude of cell contraction are significantlyincreased as well. This phenomenon was observed in both neonatal andadult cardiac myocytes, smooth muscle cells and neuron ganglia cells.These observations suggest that an electric field is able to alter thearrangement of cultured electrically-excitable cells, and improve theiralignment and confluence. The increase in cell motion as a function ofthe strength of the applied electrical stimulation during growth isdepicted in the bar graph of FIG. 6 b. There is a doubling of cellmotion seen after the application of a field strength of 5 V/cm duringgrowth.

Moreover, seeding a second time in the same culture dish we found thatthe new cells grew in alignment with the old cells, growing confluentlyinto the old cells while electric field is applied as depicted in themicrophotograph of FIG. 7. The arrangement of these cells is organizedin the same manner as in normal tissue. These cells can contractvigorously and synchronically.

In our preliminary study, we also found that the long axis of the rodshaped myocytes always grow along the direction of the electromagneticfield fringe. Additionally, if we change the direction of theelectromagnetic field, the direction of the growing cell will alsochange following the direction of the electromagnetic field fringe asdepicted in the microphotograph of FIG. 8. These observations suggestthat an electromagnetic field is able to control the direction in whichthese cells grow to allow us to generate a tissue with any cellularorientation needed. In view of the complexity of the myocardiumstructure, this feature could greatly improve the mechanical andelectrical synchronization of the transplanted cells and surroundingtissue and reduce the coupling resistance.

In the illustrated embodiment of the invention the method is implementedinto a method for cellular transplantation. After cells are transplantedinto the target organ, an electromagnetic field with an optimized fieldstrength and pulse frequency is applied to the transplanted area.

Consider three different designs for a device for applying anelectromagnetic field in any organ or tissue. The first embodiment isillustrated in FIGS. 1 a and 1 b, which conceptually illustrate anoncatheter-based device for applying an electromagnetic field totransplanted cells directly from the outside of an organ, such as heart,pancreas, nerve, brain, skeletal muscle, etc. FIGS. 1 a and 1 b show asimplified side elevation view of a human heart 10. Cells are plantedinto a target area 12 by direct injection intra-arterial infusion intothe target organ or tissue 12 during surgery. The electromagnetic fieldis applied using a non-catheter-based device. Typically, inpost-infarction patients with a low ejection fraction, cellulartransplantation into a damaged myocardium is performed during coronaryartery bypass surgery. An electrode array 14 is directly placed on thetransplanted area 12. A percutaneous wire 16 is connected to a pulsegenerator 18. The electric pulses are applied during the first week (orlonger period of time) of post cellular transplantation.

FIGS. 2 a and 2 b illustrate a catheter-based device for application ofelectromagnetic field to transplanted cells from the inside of an organcavity, such as heart, vessel, etc. If the cells are transplanted bymeans of intra-arterial infusion, without surgically exposing the organsor tissue, the catheter-based device of FIGS. 2 a and 2 b isadvantageously employed. In case of the heart, two kinds of catheters 20are used for this purpose. One catheter 20 b as shown in FIG. 2 b isused for the whole cavity and the other catheter 20 a as shown in FIG. 2a is used for a part or portion of the heart tissue. After the cells areinfused through a coronary artery into the whole or part of the heart10, catheter 20 a or 20 b is inserted into the heart cavity, usuallyeither into the ventricle or atrium.

An array of electrodes is then deployed from catheter 20 a or 20 b tocover the interior or endocardial surface of the heart chamber or aportion of the endocardium. FIG. 2 a shows catheter 20 a which includesa protection sheath 22 on its distal portion. The displacement of sheath22 relative to the distal end 26 of catheter 20 a allows the expansibledeployment of a net or resilient array 24 of interconnected electrodes.Net or resilient array 24 can be devised in any manner now known orlater devised. In the illustrated embodiment array 24 is folded insideof sheath 22 and is stretched out or deployed in a predetermined threedimensional configuration or surface by means of the biased resiliencyof a plurality of deployment wires 28, which may also carry the currentto electrodes in net or array 24 from connectors (not shown) at theproximal end of catheter 20 a. The form and mechanism by whichexpansible net or array 24 may be provided is not material to theinvention. The technology of expansible stents and the like is readilyavailable to provide a large variety of desired array shapes andqualities. Once deployed, net or array 24 is positioned against thetarget tissue area by manipulation of catheter 20 a or anchored into thetarget tissue area by conventional means or anchors which are includedas part of the structure of net or array 24.

In the case of a whole chamber deployment, net or array 24 is providedon the surface of an expansible balloon 30 as shown in FIG. 2 b at ornear the distal end of catheter 20 b. Again wiring or conductive pathsin catheter 20 b provide for the electrically connection to theplurality of electrodes from on or in the exterior surface of balloon30. The form and mechanism by which expansible net or array 24 may beprovided on balloon 30 is again not material to the invention. Thetechnology of balloon catheters is readily available to provide a largevariety of desired array shapes and qualities for a balloon-supportedand deployed net or array 24.

The catheter 20 a or 20 b is connected to a pulse generator 18 (notshown). The electric pulses are applied during the first week (or longerperiod of time) of post cellular transplantation. Fringingelectromagnetic fields are formed between the electrodes in net or array24 into the adjacent tissue as diagrammatically shown in FIG. 2 c.

FIGS. 2 d, 2 e and 2 f illustrate a catheter based device used topromote cellular transplantation in a vessel or elongate hollow organ34. In this embodiment a catheter 20 c is provided with an expansibledistal stent 32 on or in which the electrodes of net or array 24 areprovided. FIG. 2 f illustrates that after placement, stent 32 isexpanded by conventional means thereby bringing net or array 24 incontact with or at least into close proximity to the interior walls ofvessel 34. The electromagnetic field may then be applied and as shown inFIG. 2 f the cellular infusion performed through the expanded supportingstent 32 thereby exposing the endovascular tissue of vessel 34 to thefringing fields of net or array 24.

FIG. 3 illustrates an embodiment of the invention in which animplantable wireless device 36 which includes an array 24 is deployedfor repeated or long-term application of an electromagnetic field totransplanted cells from the outside of an organ, such as heart, brain,or tissue, such as nerve, skeletal muscle, etc. The wireless device 36is planted during the surgery, such as coronary artery bypass surgery asshown in FIGS. 4 a and 4 b. FIG. 4 diagrammatically illustrates a device36 which is implanted over the exterior of the entire heart, while FIG.4 b diagrammatically illustrates a device 36 which is implanted into oronto only a selected region of the heart. The electric pulses areapplied inductively by means of a broadcasting or inductive coupledpulse generator (not shown), which can be placed exterior to the chestcavity or subcutaneously implanted in the proximity of the heart. Inthis case, net or array 24 functions as a receiving antenna or inductorto the coupled electromagnetic energy from the coupled source orgenerator. The embodiment of FIG. 3 is used for long term therapy andrepeated cellular transplantation procedure into a part or whole chamberof the heart. This embodiment thus avoids repeated catheterinterventions and long-term catheterization induced infection and othercomplications.

The application of electromagnetic fields to the tissue facilitates andincreases the efficiency of the cell transplantation. Using the devicesdescribed above, the target organ and tissue can electropermeablized.Electropermeablizaton allows the infused cells to penetrate theendothelia cell layer much easier and faster in catheter mediated celltransplantation. Specially, when biologically engineered celltransplantation to whole heart is performed in cardiomyopathy and heartfailure patients, whole heart electropermeablizaton with low voltagehigh intensity pulses increases the efficiency of cell transplantation.It also facilitates cell transplantation by direct injection.

The invention overcomes a major obstacle and marks a new milestone inthe cellular transplantation field. The invention opens a new era forbiological study to explore the electromagnetic field effects on thebiologic structure and function of the human and animal cells. This newtechnology, which has never been used in existing practices, however,has a potential to greatly improve the efficacy of the celltransplantation and make it to be clinically applicable.

Many alterations and modifications may be made by those having ordinaryskill in the art without departing from the spirit and scope of theinvention. Therefore, it must be understood that the illustratedembodiment has been set forth only for the purposes of example and thatit should not be taken as limiting the invention as defined by thefollowing claims. For example, notwithstanding the fact that theelements of a claim are set forth below in a certain combination, itmust be expressly understood that the invention includes othercombinations of fewer, more or different elements, which are disclosedin above even when not initially claimed in such combinations.

The words used in this specification to describe the invention and itsvarious embodiments are to be understood not only in the sense of theircommonly defined meanings, but to include by special definition in thisspecification structure, material or acts beyond the scope of thecommonly defined meanings. Thus if an element can be understood in thecontext of this specification as including more than one meaning, thenits use in a claim must be understood as being generic to all possiblemeanings supported by the specification and by the word itself.

The definitions of the words or elements of the following claims are,therefore, defined in this specification to include not only thecombination of elements which are literally set forth, but allequivalent structure, material or acts for performing substantially thesame function in substantially the same way to obtain substantially thesame result. In this sense it is therefore contemplated that anequivalent substitution of two or more elements may be made for any oneof the elements in the claims below or that a single element may besubstituted for two or more elements in a claim. Although elements maybe described above as acting in certain combinations and even initiallyclaimed as such, it is to be expressly understood that one or moreelements from a claimed combination can in some cases be excised fromthe combination and that the claimed combination may be directed to asubcombination or variation of a subcombination.

Insubstantial changes from the claimed subject matter as viewed by aperson with ordinary skill in the art, now known or later devised, areexpressly contemplated as being equivalently within the scope of theclaims. Therefore, obvious substitutions now or later known to one withordinary skill in the art are defined to be within the scope of thedefined elements.

The claims are thus to be understood to include what is specificallyillustrated and described above, what is conceptionally equivalent, whatcan be obviously substituted and also what essentially incorporates theessential idea of the invention.

1. A method for achieving confluence and alignment of transplanted cellsin tissue comprising: transplanting cells into a selected region of thetissue; applying a pulsed electric field to the selected region of thetissue for a predetermined period of time, wherein applying the pulsedelectric field comprises exposing the selected region of the tissue to apeak electrical field strength of approximately 50 mV/cm to 5 V/cm; andrepeating application of a pulsed electric field to the selected regionof the tissue with transplanted cells disposed therein.
 2. The method ofclaim 1 where applying a pulsed electric field comprises exposing theselected region of the tissue to a pulsed electrical field with a pulserate of approximately 0.2 Hz to 20 Hz.
 3. The method of claim 1 wheretransplanting cells into a selected region of the tissue is repeatedfollowed by repeated applications of the pulsed electric field to theselected region in which repeated transplantations occur.
 4. The methodof claim 1 where the repeated applications of the pulsed electric fieldoccur over a period equal to or in excess of 24 hours.
 5. The method ofclaim 1 where the repeated applications of the pulsed electric fieldoccur over a period equal to or in excess of 7 days.
 6. The method ofclaim 1, where the tissue is selected from a group consisting of:cardiac tissue, skeletal tissue, and nerve tissue.
 7. The method ofclaim 1, where the transplanted cells are selected from a groupconsisting of adult cardiac myocytes, smooth muscle cells, neuron cells,autologous myoblasts, fetal cardiomyocytes, and allogenic fetal skeletalmyoblasts.
 8. The method of claim 1, where the tissue is located in astructure selected from a group consisting of: an organ, a vessel, amuscle, and a nerve fiber.
 9. A method for achieving confluence andalignment of transplanted cells in tissue comprising the steps of:transplanting cells into a selected region of tissue; generating apulsed electric field at the selected region of tissue; and applying thepulsed electric field to the transplanted cells in the selected regionof tissue over a predetermined time, wherein applying the pulsedelectric field comprises exposing the selected region of the tissue to apeak electrical field strength of approximately 50 mV/cm to 5 V/cm. 10.The method of claim 9 where the step of applying the pulsed electricfield comprises applying the pulsed electric field by means of anoncatheter device for applying the pulsed electric field externally toa selected region of the tissue from an nonvascular position relative tothe tissue.
 11. The method of claim 10 where the step of applying thepulsed electric field comprises a step of applying the pulsed electricfield to the selected region of tissue using an electrode array placedinto contact with the tissue in which the transplanted cells have beentransplanted.
 12. The method of claim 9 where the step of generating thepulsed electric field comprises generating the pulsed electric field fora period of time after the cellular transplantation equal to or inexcess of 24 hours.
 13. The method of claim 9 where the step ofgenerating the pulsed electric field comprises generating the pulsedelectric field for a period of time after the cellular transplantationequal to or in excess of 7 days.
 14. The method of claim 9 where thestep of applying the pulsed electric field comprises applying the pulsedelectric field to a selected region of the tissue from a vascular orinternal position relative to the tissue by means of a catheter device.15. The method of claim 14 where applying the pulsed electric field bymeans of a catheter device comprises deploying an electrode array fromthe catheter device to form an expansible net of electrodes coupled to apulse generator for contact or near contact with the transplanted cellsin the tissue.
 16. The method of claim 14 where applying the pulsedelectric field by means of a catheter device comprises deploying anelectrode array on an expansible balloon, which is inflatable tosubstantially fill an organ cavity and thereby bring the electrode arrayinto contact or near contact with the transplanted cells in the tissue.17. The method of claim 9 where the step of generating the pulsedelectric field comprises forming a fringing electric field between theelectrodes of an array into the tissue.
 18. The method of claim 17 whereforming a fringing electric field between the electrodes of the arraycomprises providing an array of electrodes having an alternating patternof electric polarity applied thereto by a pulse generator.
 19. Themethod of claim 9 where the step of applying the pulsed electric fieldcomprises forming a fringing electric field by means of an elongatecylindrical-shaped array of electrodes.
 20. The method of claim 19further comprising infusing the transplanted cells into the tissuethrough the elongate cylindrical-shaped array of electrodes.
 21. Themethod of claim 9 where the step of generating the pulsed electric fieldcomprises inductively powering an array of electrodes implanted into thetissue in proximity to the transplanted cells by inductively coupling agenerator to the array.
 22. The method of claim 9, where the tissue isselected from a group consisting of: cardiac tissue, skeletal tissue,and nerve tissue.
 23. The method of claim 9, where the transplantedcells are selected from a group consisting of adult cardiac myocytes,smooth muscle cells, neuron cells, autologous myoblasts, fetalcardiomyocytes, and allogenic fetal skeletal myoblasts.
 24. The methodof claim 9, where the tissue is located in a structure selected from agroup consisting of: an organ, a vessel, a muscle, and a nerve fiber.